1a.Objectives (from AD-416)
Objective 1: Elucidate the role of fungal extracellular enzymes as virulence factors important in postharvest decay of fresh fruit including the effect of pathogen modification of the host environment on virulence factors.

1b.Approach (from AD-416)
To elucidate the role of fungal extracellular enzymes as virulence factors in postharvest decay of apple and pear fruit, we will first compare the ability of several strains of a highly virulent and a weakly virulent pathogen of the same genus to produce extracellular enzymes (such as polygalacturonases and cellulases) during fruit infection and in response to fungal modification of the host/pathogen microenvironment. 'Golden Delicious' apple and 'Anjou' pear fruit will be infected with P. expansum or P. solitum and we will extract, purify, and characterize the extracellular enzymes produced by these organisms using standard procedures. We will develop and utilize recombinant antibodies specific for binding to and inactivating enzymes produced by the pathogens in vivo and in vitro. The antibodies will allow us to determine which specific enzymes play a major role in virulence of Penicillium species on pome fruit. Strategies incorporating recombinant antibodies against virulence factors, antioxidants known to have anti-fungal activity, and mild stress treatments will be tested for their ability to control postharvest decays of pome fruits caused by Penicillium species.

3.Progress Report
Targeting fungal virulence factors and development of novel control strategies for control of blue mold on pome fruit.

Maintaining fruit quality by inhibiting decay is crucial to prevent postharvest losses. P. expansum and P. solitum are the most destructive pome fruit pathogens causing blue mold. Polygalacturonases (PGs) are cell wall degrading enzymes that are involved in fungal virulence and aid in pathogen ingress and decay. Isolation and purification of PGs from P. expansum and P. solitum-decayed pear and apple fruit has been completed. Biochemical characterization of these enzymes and determination of their role in fruit decay may provide a foundation for development of novel disease control methods. Synthesis of a recombinant phage display library against P. expansum produced PG has been achieved and will lead to inhibition studies in the lab and eventually on apple and pear fruit surfaces. The use of recombinant antibodies to block PG activity may provide an effective and non-chemical method of preventing postharvest decay caused by Penicillium species.

4.Accomplishments
1.
Extraction, isolation, purification, and biochemical characterization of a polygalacturonase from Penicillium expansum-decayed pear fruit. Maintaining fruit quality by inhibiting decay is crucial to prevent postharvest losses. P. expansum and P. solitum are the most destructive pome fruit pathogens causing blue mold. Polygalacturonases (PGs) are cell wall degrading enzymes that are involved in fungal virulence and aid in pathogen ingress and decay. Therefore, we have extracted and purified a PG from P. expansum and P. solitum-decayed pear fruit tissue. Full biochemical characterization of the PG from P. expansum-decayed pear fruit has also been carried out. This information will be helpful to other postharvest plant pathologists in developing decay control strategies by inhibiting PGs. Ultimately, this knowledge may be applied either through genetic manipulation in the host or by identifying PG inhibitors that may be applied to the fruit surface.

2.
Production of polyclonal antisera and a recombinant phage library against polygalacturonase produced by P. expansum. It is necessary and ecologically responsible to develop alternatives to chemical control for reducing postharvest losses. In cooperation with an ARS scientist at the Appalachian Fruit Research Station, Kearneysville, West Virginia, ARS scientists in Beltsville, Maryland, have made significant progress toward the production of polyclonal antisera and a phage library against polygalacturonase from P. expansum, the causal agent of blue mold of apples. We have successfully synthesized the recombinant phage display library, completed 2 rounds of panning and identified many strong binders. Blocking polygalacturonase activity on the fruit surface using recombinant antibodies against polygalacturonase may lead to a novel control strategy to halt postharvest decay mediated by P. expansum on apple fruit. This control strategy could potentially benefit the postharvest industry by reducing dependency on chemical fungicides.